Molecular Epidemiology of Isoniazid-resistant M tuberculosis in Port-au-Prince, Haiti

Abstract Background Isoniazid-resistant, rifampin-susceptible tuberculosis (Hr-TB) is associated with poor treatment outcomes and higher rates of acquisition of further drug resistance during treatment. Due to a lack of widespread diagnostics, Hr-TB is frequently undetected and its epidemiology is incompletely understood. Methods We studied the molecular epidemiology of Hr-TB among all patients diagnosed with culture-positive pulmonary tuberculosis between January 1 and June 30, 2017, at an urban referral tuberculosis clinic in Port-au-Prince, Haiti. Demographic and clinical data were extracted from the electronic medical record. Archived diagnostic Mycobacterium tuberculosis isolates were tested for genotypic and phenotypic isoniazid resistance using the Genotype MTBDRplus assay (Hain, Nehren, Germany) and culture-based testing, respectively. All isoniazid-resistant isolates and a randomly selected subset of isoniazid-susceptible isolates underwent whole-genome sequencing to confirm the presence of mutations associated with isoniazid resistance, to validate use of Genotype MTBDRplus in this population, and to identify potential transmission links between isoniazid-resistant isolates. Results and Conclusions Among 845 patients with culture-positive pulmonary tuberculosis in Haiti, 65 (7.7%) had Hr-TB based on the Genotype MTBDRplus molecular assay. Age < 20 years was significantly associated with Hr-TB (odds ratio, 2.39; 95% confidence interval, 1.14, 4.70; P = .015). Thirteen (20%) isoniazid-resistant isolates were found in 5 putative transmission clusters based on a single nucleotide polymorphism distance of ≤ 5. No patients in these transmission clusters were members of the same household. Adolescents are at higher risk for Hr-TB. Strains of isoniazid-resistant M tuberculosis are actively circulating in Haiti and transmission is likely occurring in community settings.

Isoniazid (INH) resistance is the most common type of drug resistance in Mycobacterium tuberculosis (Mtb) among standard first-line drugs [1].Diagnostics to detect INH resistance are expensive and thus often unavailable in low-resource settings.When INH resistance is undetected and individuals with isoniazid-resistant tuberculosis (Hr-TB) are treated with drugsusceptible regimens, they experience higher rates of acquired drug resistance, treatment failure, and death compared to those with drug-susceptible tuberculosis [2,3].INH resistance is often a critical early step along the pathway to acquisition of the multidrug-resistant phenotype [4,5].
To determine the prevalence of and risk factors for Hr-TB, we retrospectively tested archived Mtb isolates from all patients diagnosed with culture-positive pulmonary tuberculosis for INH resistance during a 6-month period in 2017 at an urban tuberculosis clinic in Port-au-Prince, Haiti.

Study Setting and Population
This study was conducted at GHESKIO in Port-au-Prince, Haiti.GHESKIO is the largest tuberculosis treatment center in Haiti.Haiti comprises 10 geographic departments; the West department, which includes Port-au-Prince, has the highest population density.Based on a spatial analysis of Haiti's National TB Program surveillance data, GHESKIO diagnoses approximately 10% of all TB in Haiti and 24% of all TB diagnosed in the Port-au-Prince metro region [6].
All patients who present to GHESKIO with symptoms suggestive of tuberculosis have spot and early morning sputum examined for Mtb by the Xpert MTB/RIF (Cepheid, Sunnyvale, CA) molecular assay.If a sputum sample is positive for Mtb on Xpert, the sample then undergoes liquid culture in the BACTEC system in an on-site BSL3 laboratory.GHESKIO diagnoses approximately 1500 culture-positive tuberculosis cases annually.It serves as the main referral TB laboratory for Haiti.

Open Forum Infectious Diseases
All patients at GHESKIO between January 1, 2017 and June 30, 2017, diagnosed with pulmonary tuberculosis based on clinical symptoms, chest radiograph consistent with pulmonary tuberculosis, and positive for Mtb by the Xpert MTB/RIF molecular assay without evidence of rifampin resistance at time of diagnosis and with an archived diagnostic Mtb culture isolate were included in this study.
Persons with strains of Mtb resistant to rifampin were excluded because the epidemiology of rifampin-resistant tuberculosis is better understood in Haiti and outside the focus of this investigation.

Study Design
This was a retrospective, cross-sectional study designed in 2019 of the prevalence of isoniazid resistance among patients diagnosed with culture-positive rifampin-susceptible pulmonary tuberculosis at GHESKIO in Port-au-Prince, Haiti in 2017.GHESKIO routinely cultures all diagnostic sputum samples that are positive for Mtb based on the Xpert MTB/RIF molecular assay and routinely archives isolates from all positive cultures at −80 °C.Archived diagnostic culture isolates from patients diagnosed with pulmonary tuberculosis between January 1 and June 30, 2017, were tested for isoniazid resistance using the Genotype MTBDRplus molecular assay (Hain Lifescience, Nehren, Germany).All isolates determined to be isoniazid resistant by this molecular assay and an equal number of randomly selected isoniazid-susceptible isolates to serve as controls underwent phenotypic and genotypic drug susceptibility testing.
To confirm that the Genotype MTBDRplus assay was detecting mutations associated with phenotypic INH resistance in this population, DNA from all INH-resistant isolates and an equal number of randomly selected INH-susceptible isolates that underwent culturebased drug susceptibility testing all underwent whole-genome sequencing.DNA was extracted from all INH-resistant isolates and the same equal number of INH-susceptible isolates that underwent drug susceptibility testing and shipped for whole-genome sequencing to the Wadsworth Center of the New York State Department of Health.Clinical and demographic information were extracted from the electronic medical record.For each patient with TB, GHESKIO staff documents household contacts; this was extracted from the medical record to determine if patients were in the same household.

Laboratory Procedures
Banking of Mtb isolates: Sputum samples were adjusted to 5 mL with sterile water and decontaminated with 5 mL of NALC-NaOH (3% sodium hydroxide, 0.5 to 0.6% N-acetyl-L-cysteine, 1.47% sodium citrate).Automated liquid culture was performed using the BACTEC MGIT 960 instrument (Becton and Dickenson, Franklin Lakes, NJ, USA) in accordance with the manufacturer's instructions.The presence of Mtb in positive BACTEC cultures was confirmed with the SD-Bioline rapid MPT64 test (Standard Diagnostics, Yongin, Korea).All Mtb isolates are routinely preserved by freezing in 7H9 Middlebrook media supplemented with 15% glycerol at -80 ° C in the GHESKIO laboratory.
Molecular drug susceptibility testing: Fifty microliters of preserved Mtb isolate were transferred into a microtube with 300 µL of nuclease-free water.Bacteria were heat killed by incubation at 80 °C for 1 hour.DNA was released with 15 minutes of sonication in an ultrasound bath and the resulting crude extract was separated from debris by centrifugation and tested directly with the Genotype MTBDRplus line probe assay in accordance with the manufacturer's instructions [7].
Phenotypic drug susceptibility testing: Mtb strains found resistant to INH by the Genotype MTBDRplus assay and an equal number of randomly selected INH-susceptible strains, also based on the Genotype MTBDRplus assay, were regrown in MGIT 960 tubes and tested for resistance to 0.1 µg/mL of INH by MGIT 960 SIRE assay (Becton Dickenson) according to the manufacturer's instructions.
Isolation of genomic DNA: Frozen Mtb isolates were thawed, and 0.1 mL was inoculated into a BACTEC MGIT tube and incubated at 37 °C for 3-7 days to obtain sufficient material for genomic DNA preparation.Following incubation, 1.5 mL bacterial suspension from the bottom of the MGIT tube was transferred into a microtube and heat-killed for 1 hour at 80 ° C. Cell material was collected by centrifugation for 15 minutes at 15,000×g, then resuspended in 200 µL of the InstaGene matrix (BioRad, Hercules, USA) and incubated for 30 minutes at 56 °C.Contents of the tube were transferred into a screw-cap XXTuff reinforced microtube with 0.1-mm zirconia/silica beads (Biospec Products, Bartlesville, USA), vortexed for 10 seconds and then incubated for 20 minutes at 100 °C.Mtb cells were disrupted by bead-beating for 30 seconds at maximum speed in a D1030 BeadBug microtube homogenizer (Benchmark Scientific, Sayreville, USA).After 15 minutes' centrifugation at 15,000×g, 100 µL of supernatant containing purified genomic DNA were collected into a clean microtube.

Data Analysis
Clinical and demographic data: The association between individual-level covariates and INH resistance were first examined using a univariable logistic model.Covariates included age, sex, HIV status, prior INH use, history of TB, and neighborhood of primary residence.Variables statistically significant in univariable analysis and those judged to be clinically significant were included in a multivariable logistic model.Potential interactions between covariates in the multivariable model were also examined.
The frequency of Mtb lineages based on the Coll [9] scheme among INH-resistant and INH-susceptible strains was assessed using the chi-squared test.The distribution of the Mtb sublineages among the INH-resistant and INH-susceptible isolates were assessed using the Simpson index of diversity.The statistical significance and 95% confidence intervals (CI) for the Simpson index of diversity were estimated using the resampling approach.
Putative transmission analysis: Genetic relatedness between pairs of strains was determined by the number of SNPs distinguishing the strains across the whole genome after masking problematic genomic sites, including those containing repetitive sequences that may not evolve neutrally and may bias phylogenomic analyses.A strict distance of ≤5 SNPs as well as a less conservative SNP distance of ≤12 was used to define genetic clusters containing related strains, which may be part of a relatively recent transmission chain [19][20][21][22][23]. Pairs of strains with SNP distances less than the specified maximum SNP distance threshold were joined by an edge to construct a network showing the connectedness of the sequenced strains to infer potential transmission clusters.The network was constructed and visualized using igraph (V1.2.6) package [24] in R. Once transmission clusters were determined based on sequencing data, household contact data extracted from the medial record was examined to determine if participants represented in clusters were from the same household.

Ethical Oversight and Patient Consent Statement
This study was approved by the Weill Cornell Medicine and GHESKIO institutional review boards.It was a retrospective study of laboratory data and written patient consent was not required.

RESULTS
Between January 1, 2017, and June 30, 2017, 872 patients were diagnosed with Xpert-positive, culture-positive pulmonary tuberculosis at GHESKIO (Figure 1).Twenty-seven patients were excluded from the final study cohort because of rifampin resistance on Xpert MTB/RIF (n = 22) or no archived diagnostic culture isolate (n = 5).
The final study population included 845 patients diagnosed with Xpert-positive, culture-positive pulmonary tuberculosis without evidence of rifampin resistance.One hundred and eleven (13%) patients had HIV (Table 1).Ninety-three patients (11%) were adolescents <20 years old, and all of these adolescents were HIV negative.Of the 845 patients, 746 (89%) were presenting with a new case of tuberculosis and 99 (11%) had been previously treated for tuberculosis (it was not determined if these subsequent episodes were recurrence or relapse).

Prevalence
All isolates were screened for the presence of mutations in the 2 main molecular determinants of INH resistance by the Genotype MTBDRplus line probe assay: S315 codon of the katG gene and the mabA-inhA promoter region.Based on this assay, 65 of 845 (7.7%) patients had Hr-TB.Of 746 patients with their first episode of tuberculosis, 55 (7.4%) had Hr-TB; 10 Epidemiology of Hr-TB in Haiti • OFID • 3 of 99 patients (10.1%) presenting with a previously treated case of tuberculosis had Hr-TB.

Clinical and Demographic Covariates
In univariable models, age <20 years was the only significant covariate (odds ratio, 1.95; 95% CI, 0.96-3.69;P = .049)(Supplementary Table 1).Age <20 years, HIV status, and type of TB (new vs previously treated) were included in the multivariable model.In this model, patients aged <20 years had 2.39 greater odds (95% CI, 1.14-4.70;P = .015) of having Hr-TB compared to the reference group (new TB cases, HIV negative, aged ≥ 20 years) (Figure 2).Of note, in a subanalysis of only those with HIV, prior INH monotherapy within the context of preventive therapy among those with HIV did not impact the odds of having Hr-TB.

Phenotypic Drug Susceptibility Testing by Bactec ACTEC Liquid Culture
Of the 65 INH-resistant isolates identified by the Genotype MTBDRplus assay, 2 isolates did not grow and were not included in further analysis (Figure 1).Sixty-three INH-resistant isolates were tested for phenotypic drug susceptibility using the MGIT 960 SIRE assay in BACTEC liquid culture system.All 63 isolates (100%) were phenotypically resistant at an INH concentration of 0.1 µg/mL in liquid culture.Of the 65 randomly selected INH-susceptible isolates based on the Genotype MTBDRplus assay, all 65 were susceptible to INH at 0.1 µg/mL in the MGIT 960 SIRE assay.
Of the 65 successfully sequenced isolates in the INH-susceptible group, 1 isolate in sublineage 4.1.2.1 [9] was found to harbor a synonymous L203L mutation in the mabA gene.This mutation is known to be associated with INH resistance [25] but the Genotype MTBDRplus assay is not designed to detect it.This isolate was susceptible to INH at the 0.1 µg/mL concentration when tested in liquid culture.Following the sequencing results, this isolate was additionally tested on 7H10 Middlebrook solid media with Agar Proportion method as recommended [26].The isolate was found to be resistant to the low concentration of INH (0.2 µg/mL) but sensitive to the high concentration (1 µg/mL) of INH on solid media.Adolescents are uniquely at risk for tuberculosis because of both increased epidemiologic and immunologic susceptibility to Mtb infection [27][28][29].Studies examining the natural course of tuberculosis from the prechemotherapy age also suggest adolescence may be a high risk time for rapid progression from Mtb infection to disease onset [27,[30][31][32].Once symptomatic, they are also at risk for transmitting Mtb because of frequent delays in diagnosis, large social networks, and high number of contacts [31,[33][34][35].Younger age has been associated with Hr-TB in other settings as well [36,37].Given adolescents' younger age, Mtb strains that cause disease in adolescents most likely represent currently circulating strains, compared to adults with tuberculosis who may have been infected with Mtb decades earlier.The higher proportion of Hr-TB in adolescents in Haiti suggests that the transmission of INHr strains may be increasing.
People with HIV presenting with their first episode of tuberculosis may demonstrate a similar phenomenon because they are also at elevated risk of rapid progression from Mtb infection to symptomatic disease [38].In essence, both adolescents and people with HIV may act as "canaries in the coal mine" and may be harbingers of emerging INH resistance.Of note, there was no association between the use of INH preventive therapy and Hr-TB in our patients with HIV.
Identifying sentinel groups that may predict future prevalence of drug-resistant TB would be of great benefit for resource-limited settings, which many TB-endemic countries are.It would enable TB programs to direct surveillance efforts at a small group of patients who would provide critical data to prevent widespread transmission of certain Mtb strains.In situations of civil unrest such as that facing Port-au-Prince, this would enable limited resources to be put to their best use.
Twenty-two percent of INH-resistant Mtb isolates from patients with a new diagnosis of tuberculosis were in 5 distinct molecular clusters.These clusters were not among household contacts suggesting community transmission outside the home.This is consistent with studies demonstrating that a significant proportion of Mtb transmission occurs in community settings outside the household [39,40].A large proportion of the INH-resistant strains in our study were from the same  This study used data collected through routine medical records.Thus, the analysis of transmission clusters was based solely on sequencing data, which limits a full understanding of Hr-TB transmission in Port-au-Prince.Data on household contacts extracted from the medical record allowed for determination that no patients represented in molecular clusters were from the same household although epidemiologic links may have been missed.We included strains collected over a short time frame, which also limits the ability to fully describe the extent of Hr-TB transmission in Port-au-Prince.Treatment outcomes were outside the scope of this study and may have provided additional insight into the importance of Hr-TB detection in this population.Future research is needed to prospectively investigate epidemiologic links between Hr-TB cases and identify any changes in Hr-TB epidemiology that may have occurred since 2017, the year when the patients in this study were diagnosed.

CONCLUSION
This study demonstrates that adolescents may be sentinel groups for Hr-TB, raising concerns about an emerging epidemic of this form of drug resistance.Adolescents should be prioritized for screening for INH resistance.

Figure 3 .
Figure 3. Phylogenetic tree with lineage, phenotypic and genotypic drug resistance among isoniazid-resistant strains (A) and isoniazid-susceptible strains (B) as determined by Genotype MTBDRplus assay.
sublineage,Coll 4.3.4.1.Although our study design does not allow for strict comparison between these groups, it may suggest that certain Mtb sublineages are enriched for drug resistance.

Figure 4 .
Figure 4. Putative transmission clusters of M tuberculosis strains in Port-au-Prince, Haiti, stratified by isoniazid resistance as determined by Genotype MTBDRplus.(A) Clusters determined by a single nucleotide polymorphism (SNP) threshold of < 5. (B) Clusters determined by a SNP threshold of < 12.